Current methods for evaluating the various effects of drugs on cardiac or other biologic tissues in vitro routinely use time-consuming and technically-complex intracellular recording techniques. Further, in most cases, these evaluations are generally applied to syncytial preparations or to isolated cells one at a time.
Typically, intracellular recordings obtained in the absence and/or the presence of various substances are compared for evidence of the effects of those various substances on a muscle fiber's cardiac action potential. Among other aspects, a substance may affect a muscle fiber's cardiac action potential by delaying cardiac repolarization (manifested as prolongation of the action potential duration or APD) or accelerate cardiac repolarization (manifested as the shortening of the APD).
Unfortunately, several inadequacies exist: The current methods for obtaining these intracellular recordings are difficult to maintain; the experiments used to obtain the intracellular recordings are slow, primarily as a result of long drug equilibration times, especially for cardiac muscles; and the intracellular recordings require tissue harvests from multiple specimens to ensure adequate sample sizes. In addition, this approach does not evaluate on the potential effect of a substance to affect contractility (an inherent property of many excitable tissues) or changes in excitability (another inherent property of many excitable tissues).
Delayed cardiac repolarization is considered a surrogate marker for cardiac proarrythmia (and in particular, Torsades-de-Pointes). It has been repeatedly demonstrated that the effects of substances that delay cardiac repolarization are exaggerated during slower stimulation, an effect termed “reverse use-dependence.” Unfortunately, the effects of substances during accelerated (or irregular) pacing are typically not considered. This may be crucial in the evaluation of proarrhythmic risk associated with the rare, drug-induced polymorphic ventricular tachycardia known as Torsades-de-Pointes, as the initiating rhythm typically involves an irregular stimulation pattern or premature beats.
Integrated cellular responses, such as those provided from isolated myocytes, are preferred to evaluate the electrophysiology effects of substances on a body, as it is essentially unknown which cardiac ion channels or contractile proteins may be affected by chemicals and/or compounds under evaluation for safety or efficacy.
The ability of a living cell to respond mechanically (i.e., to either expand or contract) to a stimulus, particularly electrical stimulation, is dependent, inter alia, upon the recovery of the cell from prior electrical stimulation. In other words, expansion or contraction of a cell due to electrical stimulation is partially dependent on the quickness of a cell's “return to normal” bias, or repolarization, from a previous electrical stimulation. In the case of cardiac cells, this responsive ability is referred to as refractoriness, which is closely linked to the “cardiac action potential.”
A cell's cardiac action potential can be affected by many factors. For instance, the introduction/exposure of substances to a cell has been shown to have an effect on cardiac action potential. Some substances, like drugs and/or other chemicals, that delay repolarization and prolong the duration of the cardiac action potential, are said to prolong refractoriness. As an example, substances enhancing either the inward ionic (e.g., sodium or calcium) current can elicit increases in the cardiac action potential. In doing so, these substances limit the ability of a cell to respond to very rapid or premature stimulation. Through the use of such substances, refractoriness may be prolonged due to either (1) the reduction of outward repolarizing currents, or (2) the transient reduction and/or delaying of the recovery of channels conducting excitatory inward currents.
Similarly, substances that accelerate repolarization and shorten the cardiac action potential duration are said to shorten refractoriness. Changes in refractoriness have been linked to proarrhythmia. For example, delayed repolarization has been linked to ventricular proarrhythmia (including Torsades-de-Pointes), while shortened atrial repolarization (and refractoriness) has been linked to atrial proarrhythmia (such as atrial flutter and fibrillation) and ventricular fibrillation.
While changes in refractoriness can result from the effects on ionic currents, understanding a substance's effects on any individual ionic currents does not adequately predict effects on refractoriness, as multiple ionic currents can act in an integrated fashion to define refractoriness, and substances may affect multiple ionic currents in an undetermined manner at different concentrations. Thus, changes in refractoriness are typically evaluated in an intact, integrated cellular system (e.g., a muscle fiber). However, the direct electrophysiologic measures of changes (using microelectrode or patch electrode-based recording techniques) and the measurement of changes in refractoriness of integrated cellular systems are tedious, technically complex, and not amenable to higher throughput.
Thus, it is desirable to provide an improved method for detecting the effect of a substance on a body, which overcomes the disadvantages in the currently-used methodologies.
The invention provides for the evaluation of the effects, particularly electrophysiologic effects, of drugs on cells, particularly cardiac myocytes, without using the technically-demanding intracellular recording techniques of known methods, while requiring less specimen usage in a simpler manner and requiring minimal technical expertise. The invention also provides for the simultaneous assessment of changes in mechanical effects (particularly cardiac contractility) of test substances on cells while electrophysiologic parameters (changes in repolarization and excitability) are studied.
Further, the invention uses non-invasive, optical methods to determine responses, and the cells are evaluated under physiologic conditions. The detection schemes of the present invention are less demanding technically. Additionally, the detection schemes of the present invention are faster and more efficient than known approaches. Existing edge detection methods cannot easily be scaled to support many measurements from multiple cells in parallel. Edge detection is also problematic when edges are not well defined because of low image contrast, debris, or when an experimental flow cell chamber contains closely packed cells which may overlap partially. Finally, this approach is applicable to a variety of cell types (e.g., atrial and ventricular cardiac myocytes) and can be used for cells derived from any contractile tissue where a mechanical response is triggered or dependent on the recovery of electrical excitability.